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Biomedical subjects

R J Britten

Publications and source records attributed to R J Britten.

At least 145 records · Page 8Linked to original sources
PubMed1978

Studies on nucleic acid reassociation kinetics: V. Effects of disparity in tracer and driver fragment lengths.

Measurements are described of the kinetics of nucleic acid strand pair reassociation where the complementary strands are of different lengths and are present in different concentrations. Rate constants for the reaction of labelled fragments ("tracer") with excess complementary strands ("driver") were determined, both for driver fragment length greater than tracer fragment length and for the reverse case. Second order reactions and pseudo-first order reactions utilizing strand separated drivers and tracers were studied. The nucleic acids which served for this investigation were phiX174 DNA and RNA, plasmid RSF2124 DNA and E. coli DNA. Approximate empirical expressions relating driver and tracer fragment lengths with the observed rate constants were obtained for practical use. In long tracer-short driver reactions the observed rate constant for the tracer reaction increases proportionately with tracer length. In long driver-short tracer reactions the rate of tracer reaction is retarded. The latter result is unexpected and appears to represent a departure from standard interpretations of the renaturation reaction.

Coliphages↗
PubMed1977

Synthesis and turnover of polysomal mRNAs in sea urchin embryos.

The synthesis and turnover kinetics of polysomal mRNA have been measured in sea urchin embryos. Polysomes were isolated from stages ranging between mesenchyme blastula and late gastrula Strongylocentrotus purpuratus embryos which had been exposed to exogenous 3H-guanosine. The amount of radioactivity incorporated into messenger and ribosomal RNAs was determined separately as a function of time, and the precursor pool specific activity was measured in the same embryos. Synthesis and decay rate constants were extracted from the data by a least-squares procedure. Per embryo, the rate of mRNA synthesis was calculated to be about 0.13 pg min-1, while the rate of rRNA synthesis is about 0.022 pg min-1. The newly synthesized mRNA turns over with a half-time of 5.7 hr. The data support only a single decay rate for the mRNA, but small fractions of mRNA decaying at different rates cannot be excluded. Previous studies have shown that a minor fraction of the mRNA includes the least abundant, most highly diverse set of messages ("complex class" mRNAs). To determine whether mRNAs of the complex class are synthesized and degraded at similar rates, labeled mRNA was measured in hybrids formed in mRNA excess reactions with single copy DNA. These experiments showed that complex class mRNAs represent an approximately proportional amount of the new mRNA symthesis, and turn over at the same average rate as does the bulk of the mRNA. Most of the mRNAs in the embryo polysomes are newly synthesized, rather than maternal. This statement refers both to complex class mRNAs and to prevalent mRNAs. Considering the sequence homology between embryo and oocyte mRNAs shown earlier, these results indicate that many of the same structural genes active during oogenesis are being transcribed in embryos at these stages.

Animals↗
PubMed1977

Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.

The rate of reaction of double-stranded replicative form (RF) [3H]DNA of bacteriophage phiX174 with excess (+)strand DNA and (+)strand RNA was measured by standard methods of hydroxyapatite chromatography. The reactions follow pseudo-first-order kinetics and the observed rate constant for the RNA-DNA reaction differs less than 25% from that of the DNA-DNA reaction. The pseudo-first-order rate constants are close to the value predicted on the basis of the second-order rate constant measured in the renaturation of the double-stranded phiX RF [3H]DNA.

Coliphages↗
PubMed1977

Studies on nucleic acid reassociation kinetics: retarded rate of hybridization of RNA with excess DNA.

The rate of reaction of excess double-stranded bacteriophage phiX174 and plasmid RSF2124 DNA drivers with enzymatically synthesized asymmetric RNA tracers was measured. Other reactions were carried out with excess Escherichia coli DNA and E. coli RNA labeled in vivo. RNA and DNA fragment lengths were held approximately equal. For each case it was shown that in DNA excess the rate constant for RNA-DNA hybridization is 3- to 4.5-fold lower than that of the renaturation rate constant for the driver DNA. This retardation was also observed in pseudo-first-order hybridization reactions driven by excess strand-separated RSF2124 DNA. It was concluded that the rate constant for RNA-DNA by hybridization depends partially on which species is in excess.

DNA↗
PubMed1977

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.

Animals↗
PubMed1977

A program for least squares analysis of reassociation and hybridization data.

A computer program is described for the rapid calculation of least squares solutions for data fitted to different functions normally used in reassociation and hybridization kinetic measurements. The equations for the fraction not reacted as a function of Cot follow: First order, exp(-kCot); second order, (1+kCot)-1; variable order, (1+kCot)-n; approximate fraction of DNA sequence remaining single stranded, (1+kCot)-.44; and a function describing the pairing of tracer when the rate constant for the tracer (k) is distinct from the driver rate constant (kd): (formula: see text). Several components may be used for most of these functional forms. The standard deviations of the individual parameters at the solutions are calculated.

Computers↗
PubMed1976

Evolutionary divergence and length of repetitive sequences in sea urchin DNA.

The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease S1 from Aspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form S1 resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with S1 nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive S1 nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9 degrees C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.

Animals↗
PubMed1976

Contrasting patterns of DNA sequence arrangement in Apis mellifera (honeybee) and Musca domestica (housefly).

We have examined the organization of the repeated and single copy DNA sequences in the genomes of two insects, the honeybee (Apis mellifera) and the housefly (Musca domestica). Analysis of the reassociation kinetics of honeybee DNA fragments 330 and 2,200 nucleotides long shows that approximately 90% of both size fragments is composed entirely of non-repeated sequences. Thus honeybee DNA contains few or no repeated sequences interspersed with nonrepeated sequences at a distance of less than a few thousand nucleotides. On the other hand, the reassociation kinetics of housefly DNA fragments 250 and 2,000 nucleotides long indicates that less than 15% of the longer fragments are composed entirely of single copy sequences. A large fraction of the housefly DNA therefore contains repeated sequences spaced less than a few thousand nucleotides apart. Reassociated repetitive DNA from the housefly was treated with S1 nuclease and sized on agarose A-50. The S1 resistant sequences have a bimodal distribution of lengths. Thirty-three percent is greater than 1,500 nucleotide pairs, and 67% has an average size about 300 nucleotide pairs. The genome of the housefly appears to have at least 70% of its DNA arranged as short repeats interspersed with single copy sequences in a pattern qualitatively similar to that of most eukaryotic genomes.

Animals↗
PubMed1976

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster.

A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: () Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucletodie long fragments which also contain repetitive sequences.

Animals↗
PubMed1976

Single copy DNA and structural gene sequence relationships among four sea urchin species.

Measurements of the divergence of single copy DNA sequences among four sea urichin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60 degrees C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5 degrees C; S. franciscanus 51% and 3.5 degrees C; Lytechinus pictus 12% and 7.5 degrees C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06-0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.

Animals↗
PubMed1976

Structural gene sets active in embryos and adult tissues of the sea urchin.

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy 3H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy 3H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 X 10(6) nucleotides. In units of 10(6) nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA: 2.1 and 3.7; coelomocyte mRNA: 3.5 and less than or equal to 1.4; tubefoot mRNA: 2.7 and less than or equal to 0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and less than or equal to 0.6; exogastrula mRNA: 14 and less than or equal to 0.6. The total complexity of each mRNA polulation is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 X 10(6) nucleotides, appears to be shared by several of the tissues studied.

Animals↗